Nucleic acid has a structure in which many hydroxyl groups at specific sites of the saccharide component of nucleotide are connected through a diester group of phosphoric acid, that is, a structure in which alcoholic hydroxyl groups of nucleotide and phosphoric acid groups are sequentially bonded through ester linkages. Accordingly, the following two methods can be adopted for condensing two nucleotides according to the bonding in nucleic acid:
(a) the method in which a 3'- or 2'-phosphoric acid group is condensed with a 5'-hydroxyl group.
(b) the method in which a 3'- or 2'-hydroxyl group is condensed with a 5'-phosphoric acid group. Since the phosphoric acid group is bulky and the 5-hydroxyl group is of a primary alcohol and has a relatively high degree of freedom, and since the 3'- and 2'-hydroxyl groups are of a secondary alcohol and close to each other, the method (a) is the most advantageous of the above-mentioned two methods. The method (a) is further divided into the diester method, the triester method, the phosphite method, and the H-phosphonate method, according to the mode of condensation reaction. In view of the reaction yield, the stability of the intermediate, the rate of reaction, and the ease of the purification of the product, the triester method and the phosphite method are considered relatively advantageous. The reaction of the triester method can be expressed by the following formula (1): ##STR3##
In formula (1), B.sub.1 and B.sub.2 stand for a base component having the amino group protected, R.sub.1 stands for a protecting group for the hydroxyl group, R.sub.2 and R.sub.4 stand for hydrogen in the case of deoxyribonucleotide or a protected hydroxyl group in the case of ribonucleotide, and R.sub.3 and R.sub.5 stand for a protecting group for the phosphoric acid group.
After the above-mentioned triester-forming reaction, the protecting groups for the functional groups are removed to obtain the intended product. In general, there is no great difference in the solubility or the like of the reaction product, the unreacted substance, and the by-product, and therefore, it is not always easy separate and purify them.
Several methods have been proposed for facilitating this separation and purification by bringing about differences in the physical properties of the reaction products by using compounds having a high molecular weight as the compound forming a protecting group for the functional group. If the compound having a polymeric protecting group is solid under the reaction conditions or is obtainable as a solid after the reaction, the intended product can be easily separated and recovered by filtration and washing.
The site for forming R.sub.5 in the formula (1) is considered to be the site to which the polymeric protecting group is bonded, and use of a crosslinked polystyrene or silica gel having the functional group bonded thereto as R.sub.5 has been reported [V.A. Efimov et al., Nucleic Acid Res., 11, 8369 (1983)]. The oligonucleotide formed by bonding a polymeric protecting group mentioned above to the terminal phosphoric acid group is solid, and a series of solid-phase method techniques are established. In contrast, the method in which the condensation reaction represented by the formula (1) is carried out in a homogeneous solution is called the liquid phase method. In connection with the diester method, the use of polyvinyl alcohol [H. Scott et al., Makromolekular Chemie, 173, 247 (1973)], polyethylene glycol [H. Kosler, Tetrahedron Letters, No. 16, 1535 (1972)], polystyrene [H. Hayatsu, H.G. Kohorana, J. Am. Chem. Soc., 88, 3182 (1966)], and a vinyl alcohol/polyvinylpyrrolidone copolymer [H. Seliger, G. Aumamann, Tetrahedron Letters, No. 31, 2911 (1973)] as the compound for forming a polymeric protecting group has been proposed. When these solvent-soluble polymers are used, the method is regarded as a liquid-phase method, but in this case, it is not easy to separate and purify the reaction product.